Bone is a mineralized tissue of complex chemical and cellular composition which is continuously remodelled throughout the mammalian life span. Underlying the remodelling process are cells of the osteoblast lineage which participate in bone formation, and cells of the osteoclast lineage which participate in bone resorption. These two types of cells are known to originate from distinct early progenitor cells, i.e. stem cells, which differentiate along separate pathways into mature and functional cells, in response to such endogenous mediators as systemic hormones, cytokines and growth factors.
Some of the molecules that are presumed to control biological processes essential to bone development and repair have been isolated and biochemically identified, and recombinant DNA technologies have been applied to produce relatively large quantities of those having a protein-based structure. These advances are expected ultimately to provide physicians and surgeons with defined therapeutic compositions that can be administered in clinical situations when accelerated healing, sustained repair or reconstruction of skeletal and cranio-maxillofacial defects are desired, e.g. union of fractures caused by trauma or as a consequence of osteoporosis, autogenous and allogenic bone grafts, plastic surgery, and correction of congenital deformities.
Many of the compounds having a beneficial effect on bone development are proteins and glycoproteins that can be identified in or were isolated originally from extracts of demineralized i.e. acid-treated mammalian bone. These may be categorized into three distinct classes.
One class of molecules having an effect on bone cell activity includes mitogenic substances which for the most part were known previously to have a mitogenic effect on cells types other than bone cells, such as fibroblasts. Among the molecules in this class are polypeptides that exhibit heparin-binding affinity, such as acidic fibroblast growth factor (15-18 kDa) and basic fibroblast growth factor (18-22 kDa) as well as platelet-derived growth factor (28-31 kDa) and a high molecular weight (83 kDa) isoform of insulin-like growth factor II originally called skeletal growth factor.
A second class of osteogenic molecules includes cartilage-inducing factors, designated CIF-A (26 kDa) and CIF-B (26 kDa) which are identical with transforming growth factors TGF-B1 and TGF-B2. It has been demonstrated that TGF-B2 is effective in promoting bone mass increases in several animal models, but only after repeated (daily) injections of doses in excess of 250 ng. Daily injection is also required to attain increased bone mass when the osteogenic factor (1.5 kDa, pI&gt;11) disclosed in Canadian patent application 2,010,660 is employed.
A third class of osteogenic molecules includes several related members of a family of glycosylated proteins characterized by: a dimeric structure; a molecular weight in the 25 to 30 kDa range; the presence of several interchain disulfide bonds essential to bioactivity; moderate affinity for heparin; substantial amino acid homology with species of TGF-B; and the ability to initiate bone formation at non-bone sites when combined with a bone-compatible matrix and administered to rats. Included in this class are the several species of "bone morphogenetic protein" (28-30 kDa). Also included are substances that are less well characterized but share at least one characteristic with members in this class, such as the osteogenic factor (22-24 kDa) disclosed in U.S. Pat. No. 4,804,744.
As mentioned above, these osteogenic substances have for the most part been detected in, and isolated from, extracts of previously demineralized mammalian bone fragments. In the extraction protocol commonly applied to the demineralized material, use is made of extractive solutions that include a dissociating agent such as urea or guanidinium hydrochloride, to assist in separation of macromolecular aggregates, and include a combination of inhibitors of proteolytic activity to reduce digestion of the protein component released during extraction. An alternative approach to generating mammalian bone extracts as described in U.S. Pat. No. 3,458,397, in which fragmented bone is exposed to both a strong demineralizing acid and to the protein-digesting agent pepsin, has lead to the isolation of osteogenic material that induces remarkable increases in bone growth.
According to U.S. Pat. No. 3,458,397, osteogenic material is provided when acid/pepsin-treated calf bone extracts are precipitated in ethanol and then stripped with water to yield the insoluble component. As reported by Tornberg et al in Clinical Orthopaedics and Related Research, 1977, 129:305-312 and later in the same journal by Clark et al, 1988, 237:226-235, this osteogenic material results in a dramatic increase in bone weight when administered, in a phosphate-buffered saline vehicle, at the bone surface. A single injection of a 6 mg dose of the osteogenic material was found to induce an increase in the dry weight of rat limb bone of up to 30% relative to the untreated contralateral limb, with the maximum effect being realized at about 7 days after injection.
Though the effect on bone growth induced by the osteogenic material is a desirable one, and is valuable particularly in the context of fracture repair, the crude state of the material makes it inappropriate for use as a human therapeutic.
It is an object of the present invention to provide an isolated osteogenic factor.
It is another object of the present invention to provide an osteogenic composition useful to induce bone growth.
It is another object of the present invention to provide a method for inducing bone formation in a mammal.